实验性乳腺肿瘤细胞系脂肪酸合酶的研究

Randolph A Hennigar, Mildred Pochet, Dirk A Hunt, Aron E Lukacher, Virginia J Venema, Elizabeth Seal, Mario B Marrero
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引用次数: 21

摘要

脂肪生成酶脂肪酸合成酶(FAS)在各种人类原发性癌症和某些人类癌细胞系中升高。FAS在人类肿瘤中的过表达具有临床意义,因为它与肿瘤侵袭性和潜在的化疗干预有关。本研究对正常小鼠乳腺上皮细胞(NMuMG)和由啮齿动物多瘤病毒(Py)或小鼠乳腺肿瘤病毒(MMTV)诱导的乳腺肿瘤细胞系中的FAS进行了检测。Western blot结果显示,py转化的A1-1和T1中FAS含量高于NMuMG或mmtv转化的Mm5MT、RIIIMT和MMT060562。这些数据表明,Py转化介导的信号事件可能会增加细胞中FAS的数量。虽然FAS含量在A1-1和T1处理时达到了相近的水平,但比活性差异显著,T1处理的酶活性是A1-1的3倍。同样,尽管酶含量相似,但NMuMG中的FAS活性比mmtv转化系高约0.5倍。采用抗磷酸氨基酸抗体进行免疫沉淀研究,然后用抗fas抗血清进行免疫印迹分析(反之亦然),以表征酶的构成磷酸化状态。在来自T1和NMuMG的活性较高的FAS中检测到磷丝氨酸和磷苏氨酸残基,而在来自Mm5MT和A1-1的活性较低的FAS中未检测到磷丝氨酸残基。磷酸化FAS的发现表明,这种酶可能比以前认为的对脂肪形成有更直接的控制。高剂量(10−4 M)地塞米松诱导NMuMG和mmtv转化细胞株中FAS含量和活性升高,但对py转化细胞无影响。较低浓度(10 - 8和10 - 6 M)的地塞米松也激活了FAS,但没有伴随蛋白含量的升高,这与FAS的磷酸化形式一致。最后,用FAS抑制剂cerulenin处理细胞系:几乎所有的乳腺癌细胞系在明显低于正常细胞系的药物剂量下都受到生长抑制,这表明FAS对肿瘤细胞的生存能力比正常细胞发挥更大的作用。用棕榈酸酯(FAS的主要最终产物)预处理后,蓝绿素仅能轻微地挽救A1-1细胞免受生长抑制,而用油酸酯(由棕榈酸合成的单不饱和脂肪酸)预处理可协同蓝绿素的细胞毒性作用。
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Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10−4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10−8, 10−6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.

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